TWI527595B - An inhibitor of a low degree of collagen production, a skin external composition containing the same, and a cosmetic product - Google Patents

Description

Collagen produces a low-level inhibitor, a skin external composition containing the same, and a cosmetic Cross-references to related applications

This application is based on the Japanese Patent Application No. 2009-148772 (Application Date: June 23, 2009), which is hereby incorporated by reference, and claims the benefit of priority, and The overall disclosure is included here. .

Technical field

The present invention relates to an inhibitor of collagen production of dermal fibroblasts using an enzyme decomposition product of bird's nest as an active ingredient. More specifically, the present invention relates to a collagen production inhibitor which inhibits the production of type I collagen which occurs in skin dermal fibroblasts by ultraviolet irradiation. Further, the present invention relates to a skin external composition and a cosmetic comprising an enzyme decomposition product of bird's nest containing the above-mentioned active ingredient.

Background technique

Skin problems such as dry skin, wrinkles, sagging skin, and poor elasticity are especially valued and cared for by women. These skin problems are usually thought to be caused by environmental factors, ageing factors such as ageing, and even psychological factors such as stress. In particular, environmental factors that have adversely affected the skin caused by the increase in ultraviolet rays and the drying of the living environment accompanying the widespread use of air conditioners in recent years are increasing.

The skin tissue consists of epidermis, dermis, and subcutaneous tissue. The dermis contains a lot of collagen and hyaluronic acid, which is closely related to the water retention and elasticity of the skin. Collagen and hyaluronic acid are known to be produced by dermal fibroblasts present in the dermis.

Collagen is said to account for about 30% of the protein in the body, 70% of which is found in the dermis. Collagen in the dermis contains type I collagen and type III collagen, but mainly type I collagen.

In the past, in order to improve the skin's elasticity, luster, and wrinkles, the collagen-added cosmetics were used in a non-external manner, and the collagen was directly added to the skin. However, when collagen is added by external means, it is difficult to increase the collagen of the skin dermis, so it is difficult to increase the elasticity and gloss of the skin and prevent wrinkles.

If the skin is exposed to light containing ultraviolet light, it is known that most of the collagen in the extracellular matrix of the dermis will be significantly reduced, and the fiber bundles will be irregularly arranged. Therefore, the increase in the amount of collagen in the dermis contributes to the improvement and prevention of wrinkles (Non-document 1 "Experimental method for the development of functional cosmetic materials" (Sui Xuan Xuer), issued in 2007, CMC Publishing, 45~47 pages). In addition, it is also revealed that the ultraviolet light (UVB) irradiation will greatly reduce the collagen synthesis of dermal fibroblasts. Among them, the type I collagen synthesis promoter is exemplified by a vitamin C inducer, a whey component, and the like.

In addition, the literature (Akiko Kojima-Yuasa, et al., Fragrance Journal, April, 2009, No. 346 (Vol. 37, No. 4), pp. 103-) discloses inhibition of reduction in collagen proliferative ability. It helps to improve the improvement of QOL, and the decrease in the amount of collagen in the skin is the cause of wrinkles and sagging.

Therefore, the reduction or reduction of the collagen synthesis of the dermal fibroblasts which are irradiated with ultraviolet rays is suppressed or the prevention or improvement of the elasticity and gloss of the skin which may accompany the reduction of collagen which may occur on the skin after exposure to ultraviolet rays or the wrinkles. From the point of view of prevention or improvement, it is very important.

However, heretofore, there have been various attempts to promote the proliferation of type I collagen produced by dermal fibroblasts, such as JP-A-2008-105984 and WO2004/085429, but as far as the present inventors are concerned, The phenomenon of directly inhibiting or improving the collagen synthesis of dermal fibroblasts irradiated with ultraviolet rays itself, and inhibiting the change of collagen in the dermis has not been reviewed.

The bird's nest swiftlet is made up of its own saliva, which is known to contain a large amount of glycoprotein and almost no lipid. Bird's nest has been used in Chinese cuisine as a food ingredient, and it is also a raw material for traditional Chinese medicine. In recent years, health foods such as bird's nest have been developed, and applications for external use such as external preparations and cosmetics are still less well known.

A cosmetic agent for oral use using an enzyme decomposition product of bird's nest has been disclosed in Japanese Laid-Open Patent Publication No. 2003-95961. However, only oral use is disclosed therein, and no disclosure or suggestion is made as to the possibility of use for external use. Moreover, the effect is that the moisturizing effect of the skin and the realization of the delicate skin of the skin are not disclosed in the ability to promote the elasticity and luster of the skin and to improve the production of type I collagen of wrinkles, and it is not exposed to ultraviolet rays. The inhibition of the reduction in collagen synthesis of dermal fibroblasts is not disclosed or implied.

In WO 99/022709, an aqueous solvent extract comprising bird's nest, in particular a hot water extract, has been disclosed. The cosmetic can be used for skin care purposes and is used for external use. Moreover, it is also disclosed that the aqueous extract of bird's nest has the collagen synthesis promoting effect of skin cells. However, the extract used in the bird's nest is extracted by hot water extraction to remove the solid portion, and the bird's nest enzyme decomposition product which is directly used for the enzyme treatment of the bird's nest as a whole is obviously different. Further, there is no disclosure or suggestion regarding the suppression of the reduction of collagen synthesis by ultraviolet-irradiated dermal fibroblasts.

Summary of invention

The present inventors have unexpectedly discovered that the enzyme decomposition product of bird's nest has an effect of effectively suppressing the decrease in the synthesis of type I collagen which is visible in the skin dermal fibroblast which is irradiated with ultraviolet rays. The present invention is based on the foregoing findings.

Further, the present inventors have also found that an enzyme decomposition product of bird's nest has an excellent proliferation promoting effect of type I collagen in skin dermal fibroblasts.

Accordingly, an object of the present invention is to provide an excellent inhibitor of collagen production in dermal fibroblasts which can be efficiently produced and which is highly safe, and an external preparation and a cosmetic containing the former.

The collagen-lowering inhibitor of the present invention is an inhibitor of an enzyme decomposition product of bird's nest, which inhibits the inhibitory effect of dermal fibroblasts on collagen production due to ultraviolet irradiation.

According to a preferred embodiment of the present invention, the enzyme decomposition product is a decomposition product of a protease.

According to a further aspect of the present invention, the molecular weight of the enzyme decomposition product is 70 to 200,000.

According to another more preferred aspect of the present invention, the enzyme decomposition product has an average molecular weight of from 1,000 to 50,000.

According to a preferred aspect of the present invention, the collagen-lowering inhibitor of the present invention inhibits the production of type I collagen which occurs in skin dermal fibroblasts due to ultraviolet irradiation.

According to another aspect of the present invention, there is provided a skin external composition comprising the collagen-lowering inhibitor of the present invention. The composition is preferably used for skin care before or after exposure to ultraviolet rays, and is preferably used for preventing or improving skin elasticity and gloss which occur in ultraviolet-irradiated skin and preventing or improving wrinkles.

According to still other preferred aspects of the present invention, the external composition for skin of the present invention is a cosmetic.

According to another aspect of the present invention, there is provided a use of an enzyme decomposition product of bird's nest for producing an inhibitor of collagen fibrosis caused by ultraviolet irradiation of a mammal including a human. According to another aspect of the present invention, there is provided a use of an enzyme decomposition product of bird's nest for producing a skin external composition for skin care before or after exposure to ultraviolet rays.

According to still another aspect of the present invention, there is provided a method comprising: applying an effective amount of the enzyme decomposition product of the active ingredient of the present invention to the skin of a mammal including humans, and inhibiting the dermal fibroblast factor Ultraviolet irradiation causes the production of collagen to be low. According to another aspect of the present invention, there is provided a method of skin care comprising: applying an effective amount of an active ingredient of the present invention to a mammal, including a human, on the skin before or after exposure to ultraviolet light. Similarly, a method can be provided which comprises: applying an effective amount of the active ingredient of the present invention to the skin of a mammal including humans; and improving the elasticity and gloss of the skin which occurs in the ultraviolet-irradiated skin, And prevent or improve wrinkles.

According to the present invention, the use of an enzyme decomposition product of bird's nest as an active ingredient can effectively inhibit and improve the occurrence of collagen which may occur in human skin dermal fibroblasts. In particular, the production of type I collagen which is observed in ultraviolet ray-exposed skin dermal fibroblasts is low, and an effective inhibition or improvement effect can be obtained. Further, the collagen of the dermal fibroblasts has a low inhibitory effect, and when the enzyme decomposition product is used for external use or cosmetic use, the elasticity of the skin accompanied by the collagen production of the fibroblast can be improved (elasticity). ) and the reduction of skin gloss, thereby preventing or improving wrinkles.

In particular, the above effects can improve the skin of the skin after the sun is reduced in the state of collagen synthesis. Thus, according to the present invention, an increase in the QOL of the consumer can be achieved. Further, the enzyme decomposition product used is also known as the bird's nest of the foodstuff, so that the safety is high and the consumer acceptance is high. Further, since the enzyme decomposition product can be obtained by directly performing enzyme treatment on the bird's nest, it can be efficiently manufactured and the manufacturing cost can be reduced.

Simple illustration

Figure 1 is a graph showing the results of determination of the amount of citric acid in a bird's nest sample.

Figure 2 is a graph showing the results of the measurement of the amount of protein in the bird's nest samples.

Fig. 3 is a graph showing the results of measurement of the bird's nest enzyme decomposition product 1 by GPC-HPLC.

Fig. 4 is a graph showing the results of measurement of the bird's nest enzyme decomposition product 2 by GPC-HPLC.

Fig. 5 is a graph showing the results of measurement of the ability to promote collagen production.

Detailed description of the invention Active ingredients

The collagen-lowering inhibitor of the present invention, as described above, is an enzyme decomposition product of bird's nest as an active ingredient.

The raw material of the enzyme decomposition product is the nest of the swallow that forms its own saliva into a nest. The composition contains a large amount of glycoprotein and contains almost no lipid. Here, the swiftlet refers to the swallows under the genus Collocalia of Apodidae, such as Apodidae Collocalia francica and Apodidae Collocalia. Salanga), Apodidae Collocalia fuciphaga, Apodidae Collocalia inexpectat, Apodidae Collocalia vestita, Apodidae Collocalia esculent, Dajinsi Swallow (Apodidae Collocalia maximus) and the like.

In the present invention, commercially available products can be used for the bird's nest. Generally, the bird's nest that is commercially available is formed by removing only the dirt such as swallow hair and feces, and the person who has been cleaned by collecting the debris of the bird's nest and repeatedly bleaching and washing, including various types, but the bird's nest used in the present invention is suitable. Use bird's nest that has not been cleaned and bleached before pretreatment.

The enzyme decomposition product of the bird's nest refers to the decomposition of the bird's nest by the enzyme reaction, and can be obtained by decomposing the bird's nest or its treatment with a protease or a compound enzyme containing a protease. Here, the enzyme used in the decomposition treatment is preferably a protease, and trypsin is more preferable.

The enzyme decomposition products of bird's nest can usually be heat-sterilized and then treated with enzymes in water. The conditions of the sterilization treatment can be appropriately set with reference to the conventional heat sterilization conditions, and the conditions for the enzyme treatment can be appropriately set depending on the type of the enzyme to be used and the state of the bird's nest.

More specifically, the enzyme decomposition product of bird's nest can be prepared according to the following procedure.

For example, the ground bird's nest is added with 10 to 50 times its mass and expanded, and then heat-sterilized at 60 to 130 ° C for 5 seconds to 30 minutes. Then, directly add the right amount of enzyme, and carry out the enzyme reaction for 0.5 to 48 hours at the optimum pH and optimal temperature of the enzyme. After the completion of the enzyme reaction, the enzyme is deactivated by heat treatment, and the reaction solution is filtered to remove insoluble matter, thereby obtaining an enzyme decomposition product of bird's nest. Further, the above-mentioned enzyme decomposition product may be dried into a powder form as needed. Further, in each of the above procedures, operations such as pH adjustment, decolorization, and deodorization may be appropriately performed.

In the case of enzyme decomposition of the treatment of bird's nest, he added 10 to 100 times the mass of the ground bird's nest with a particle size of 2 mm or less and 150 μm or less, and was allowed to stand at 1 to 60 ° C. After being stirred or stirred for 0.5 to 48 hours, it is sterilized by heat treatment at 60 to 130 ° C for 5 seconds to 30 minutes, and filtered as needed to prepare a solution, which is then treated with the same enzyme as above. Produce an enzyme decomposition product of bird's nest.

The enzyme decomposition product is the longer the reaction time of the enzyme, and the higher the degree of protein decomposition, the more the enzyme decomposition product containing the lower molecular protein. In the present invention, the enzyme reaction time is 0.5 to 48 hours, and 3 to 36 hours is more preferable, and the optimum degree is about 24 hours, and it is preferable to use an enzyme decomposition product after the enzyme reaction is sufficiently performed.

The molecular weight of the enzyme decomposition product of the bird's nest thus obtained is preferably 70 to 200,000, and more preferably 70 to 180,000. The molecular weight of 70~150,000 is excellent, and 70~120,000 is the best.

Further, the average molecular weight (weight average molecular weight) of the enzyme decomposition product of the bird's nest is preferably from 300 to 100,000, and more preferably from 500 to 70,000. The average molecular weight of 700~60,000 is very good, and the best of 1,000~50,000.

In addition, if the above-mentioned operating conditions are appropriately changed for the bird's nest enzyme decomposition product, or the conventional purification or classification method is applied, the lower limit of the average molecular weight may be 5,000 or more, and more preferably 10,000 or more, and 15,000 or more. Good, more than 20,000 is the best.

Skin external composition

The collagen production lowering inhibitor of the present invention can be used as it is, but it can also be used as an additive in various skin external compositions such as cosmetics, pharmaceuticals, and quasi-drugs, thereby producing collagen having dermal fibroblasts. A composition that has a low inhibitory effect. The prepared composition can be effectively used for skin care, and is preferably used for skin care before or after exposure to ultraviolet rays, for improving the elasticity and gloss of the skin caused by ultraviolet radiation, and for preventing wrinkles or Better is better.

The enzyme decomposition product of bird's nest of the active ingredient of the present invention has an activity of effectively reducing the production of type I collagen which is visible in human skin dermal fibroblasts which are irradiated with ultraviolet rays (test results of the examples described later).

Therefore, in the present invention, the hypothetical inhibitory activity of collagen is intended to inhibit or ameliorate the decrease in the ability of collagen production which may occur in fibroblasts of human skin dermis, thereby suppressing or improving humans thereby. The phenomenon of the amount of collagen in the dermis of the skin. By inhibiting or improving the amount of collagen in the dermis, the wrinkle can be improved and prevented, and the skin elasticity (elasticity) and skin gloss can be prevented or reduced due to the low collagen production, which is well known to those skilled in the art. It can be easily understood (such as the aforementioned document ("Experimental method for the development of functional cosmetic materials" (Supervisor of 芋皮玄尔), issued in 2007, CMC Publishing, pp. 45~47 (Non-Patent Document 1)) This point has been clearly stated in the literature (Akiko Kojima-Yuasa, et al., Fragrance Journal, April, 2009, No. 346 (Vol. 37, No. 4), pp. 103-(Non-Patent Document 2)) and the like. ).

In particular, the above effects can be said to improve the skin in which the collagen synthesis is reduced in the skin after sun exposure. Therefore, the active ingredient of the present invention has confirmed that the collagen having the dermal fibroblasts has a low inhibitory activity, so that the active ingredient of the present invention contributes significantly to skin care, and in particular, the active ingredient of the present invention is significantly useful for receiving ultraviolet rays. Skin care before or after exposure is more suitable for the improvement of the elasticity and gloss of the skin caused by UV-irradiated skin, as well as the prevention or improvement of wrinkles. Further, the active ingredient of the present invention contributes to the improvement of the elasticity of the skin caused by aging, the prevention of wrinkles, and the like.

In addition, in this specification, the "prevention or improvement" of the skin and the skin such as wrinkles, such as the reduction of elasticity and gloss of the skin, includes the regulation of the state, the delay of the deterioration, the reduction, the prevention of seizures, the prevention of recurrence, and the inhibition. The meaning inside.

Therefore, as described above, according to another aspect of the present invention, a skin external composition constituting the collagen-containing hyposuppressive agent of the present invention can be provided. The external composition for skin is preferably provided as a pharmaceutical, quasi-drug or cosmetic, and is more preferably provided as a cosmetic.

In the skin external composition (especially cosmetic) of the present invention, the content of the enzyme decomposition product (dry matter conversion) of the bird's nest of the active ingredient is not particularly limited, and varies depending on the form of the composition, but is generally 0.001 to 20 as a solid portion. The weight percentage is preferably in the range of 0.005 to 5% by weight, and can be appropriately changed depending on the form of the composition.

Alternatively, the content of the bird's nest enzyme decomposition product (dry matter conversion) of the external composition for skin of the present invention is preferably 0.01 mg or more per day on the skin per square centimeter, and preferably 0.01 mg to 2 mg.

The skin external composition of the present invention may further contain other optional components insofar as the skin external composition of the present invention contains an active ingredient and does not impair the inhibitory effect of the collagen production of the dermal fibroblasts. Any of the above components may be various components such as a skin external preparation and a cosmetic, and may, for example, be a moisturizer, a lower alcohol, a polyvalent alcohol, a sugar, a surfactant, a buffer, an emulsifier, a stabilizer, or a tackifier. , antioxidants, antiseptic antibacterial agents, chelating agents, pH adjusters, perfumes, pigments, UV absorbers, UV scattering agents, vitamins, amino acids, anti-inflammatory agents, water, peptides, sugar alcohols, enzymes, Plant extracts, antioxidants, talcum powder, clay, pollen, pearl powder, etc.

Here, the humectant may, for example, be an acid mucopolysaccharide such as polyethylene glycol, polypropylene glycol, glycerin, propylene glycol, sorbitol, hyaluronic acid or chondroitin sulfate, amino acid, collagen, elastin or the like.

The lower alcohol may, for example, be ethanol, propanol, isopropanol or the like.

The polyvalent alcohol may, for example, be glycerin, pentaerythritol, diisoamyltetraol, ethylene glycol, propylene glycol, polypropylene glycol, 1,3-butylene glycol or the like.

The sugar may, for example, be an inducement of sugars such as glucose, maltose, lactose, D-glucuronic acid, sucrose, D-sorbitol, sorbitol anhydride, cellulose, starch, oligosaccharides, polysaccharides, and the like. body.

The composition for external use on skin of the present invention can be appropriately formulated into a collagen-producing inhibitor containing bird's nest enzyme decomposition product and any of the above-mentioned components by a known method to form a lotion, lotion, cream, moisturizer, face lotion, makeup Forms of conventional products such as water, facial cleanser, body lotion, body cream, etc. The skin external composition of the present invention can further provide hair cosmetic products such as foundations, lipsticks, eye shadows, and blushes, sunscreen products, deodorant cosmetics, and other cosmetic products such as shampoos, moisturizers, and styling products. Uses of skin cleansers and bathing baths for bathing agents, etc.

According to another aspect of the present invention, as described above, the use of an enzyme decomposition product of bird's nest can be provided for the purpose of producing collagen fibroblasts of mammals including humans, which are caused by ultraviolet light irradiation and inhibiting collagen production. Agent. Here, the inhibition of collagen production caused by ultraviolet irradiation by dermal fibroblasts is preferably suppressed by the decrease in type I collagen which occurs in skin dermal fibroblasts caused by ultraviolet irradiation.

According to another aspect of the present invention, as described above, the use of an enzyme decomposition product of bird's nest can be provided for the purpose of producing a skin external composition for skin care before or after exposure to ultraviolet rays. Here, the external composition for skin is preferably used for the reduction of skin elasticity and gloss which occurs in the skin which is irradiated with ultraviolet rays, and the prevention or improvement of wrinkles. Further, the composition for external use of the skin is preferably a cosmetic.

According to another aspect of the present invention, as described above, there can be provided a method comprising the step of using an effective amount of an enzyme decomposition product of the active ingredient of the present invention on the skin of a mammal including a human, thereby inhibiting the dermis The production of collagen due to ultraviolet irradiation of fibroblasts is low. In addition, the "effective amount" here means a sufficient amount which can exert the inhibitory activity of the production of collagen in a predetermined field in the body by use. Further, the inhibition of the production of collagen due to ultraviolet irradiation by the so-called dermal fibroblasts is preferably such that the type I collagen produced in the dermal fibroblasts due to ultraviolet irradiation is suppressed.

Further, according to still another aspect of the present invention, a skin care method comprising a method of using an effective amount of the active ingredient of the present invention on a skin before or after exposure to ultraviolet rays of a mammal including a human can be provided. Similarly, a method of using an effective amount of the active ingredient of the present invention for the skin of a mammal including a human, and improving the elasticity and gloss of the skin, and preventing or improving wrinkles can be provided. In addition, the so-called "effective amount" refers to the sufficient amount required to achieve the desired effect.

In addition, the values of the terms "about" and "degree" are used in this specification to include the meaning of the changes that can be tolerated by the skilled person in the industry before the purpose of setting the value. For example, it means that the change within 20% of the predetermined value or range is allowed, and preferably within 10%, and preferably within 5%.

Example

The present invention will be specifically described with reference to the following examples, but the present invention is not limited by the following examples.

(1) Modulation of bird's nest samples (1-1) Bird's Nest Enzyme Decomposition 1

The commercially available unbleached bird's nest is ground by a mill to obtain a bird's nest powder having a mesh size of 100 μm or less, and about 50 times (mass) of water is added to the bird's nest powder at 5 ° C. After allowing to expand for 20 hours, heat sterilization treatment was carried out at 121 ° C for 15 minutes.

After the obtained treatment liquid was cooled, the pH was adjusted, and 2% by mass of the protease-containing enzyme (trade name "Pancreatin F", manufactured by Amano Pharmaceutical Co., Ltd.) was added to the bird's nest powder, and the reaction was carried out at 45 ° C for 3 hours. .

After adjusting the above-mentioned enzyme reaction solution to pH 7.0, the enzyme was inactivated by heating at 90 ° C for 5 minutes, and the filtrate was recovered by filtration. After concentrating the above filtrate, it is freeze-dried to obtain "bird's nest enzyme decomposition product 1"

(1-2) Bird's Nest Enzyme Decomposition

The process was changed to the same as the enzyme decomposition product 1 of the above (1-1) except that the time of the enzyme reaction was changed to 24 hours, and the "bird's nest enzyme decomposition product 2" was obtained.

(1-3) Bird's Nest Hot Water Extract (Comparative Example)

The commercially available unbleached bird's nest (dried product) was ground by a mill, and the ground material was added to 1000 ml of distilled water, followed by heating and reflux for 2 hours. After heating to reflux, it was allowed to stand still, and then the supernatant was separated and filtered to recover the filtrate. The residue was further added to 1000 ml of distilled water in the same manner as described above, and after re-extraction treatment by heating under reflux, the solid residue was filtered and removed, and the filtrate was recovered. The obtained filtrate (extract solution) was concentrated together under reduced pressure, and the obtained concentrate was freeze-dried to obtain a yellow-white "swallow's nest hot water extract" (Comparative Example).

(2) Analysis of bird's nest samples

The amount of citric acid and the amount of protein in each of the prepared bird's nest samples (bird's nest enzyme decomposition product 1, bird's nest enzyme decomposition product 2, and bird's nest hot water extract) have been determined as follows.

The amount of citric acid was determined by high-speed liquid chromatography after the acidic hydrolysis of each sample to determine the free N-acetylproline.

The amount of protein was determined using Bio Rad's Protein Assay Kit based on the Brad ford method.

The results are shown in Figures 1 and 2.

Next, in order to measure the molecular weight and distribution of the protein contained in the bird's nest enzyme decomposition product 1 and the bird's nest enzyme decomposition product 2, GPC analysis was carried out under the following conditions.

[HPLC measurement conditions]

‧Tube: Shodex Asahipak GS320HQ (ψ7.6×300mm)

‧column temperature: 35 ° C

‧Mobile phase: 50mM CH ₃ COONH ₃ pH6.7

‧Flow rate: 0.4ml/min

‧ sample injection amount: 10μl

‧Detector: UV spectrometer (measured by UV220nm)

[Molecular Weight Marker Used]

‧BSA MW66,000

‧Gly-gly GH132.1

‧Three peptides (Gly-gly-gly) MW189.17

‧L-Asparaginic acid MW133.1

‧DL-Asparaginic acid MW150.1

‧N-Acetylneuraminic acid MW309.3

The measurement results are shown in Figures 3 and 4.

Further, the molecular weight of the protein contained in the bird's nest enzyme decomposition product has been evaluated using the Western blot method. Specifically, the amount of protein in the decomposition of the bird's nest enzyme is confirmed by following the above-described measurement method. The protein derived from the bird's nest enzyme decomposition product (100 μg) was separated by 10 to 15% of polyacrylamide using SDS-PAGE. The protein dispersed in the colloid was transferred to a lmmunoblot PVDF membrane (Polyvinylidene difluoride, available from Bio-Lad Lab). The PVDF membrane was blocked for 1 hour in PBS containing 3% skim milk and 0.1% Tween-20, and visually judged.

Further, when evaluating the molecular weight of a protein, reference has been made to the information disclosed in the well-known literature (Guo, C. T., Suzuki Y., et al., Antiviral Res. 70, pp. 140-146 (2006)).

From the above results, it is understood that the molecular weight of the protein contained in the bird's nest enzyme decomposition product is in the range of about 70 to 120,000 (in addition, depending on the evaluation conditions, the upper limit is up to 200,000).

Further, from the results obtained, it was found that the average molecular weight of the protein of the bird's nest enzyme decomposition product 1 was about 55,000. Further, it is known that the average molecular weight of the protein of the bird's nest enzyme decomposition product 2 is about 33,000.

(3) The inhibitory effect of the low production of collagen in dermal fibroblasts caused by ultraviolet irradiation

Normal human skin dermal fibroblasts (KF-4109, obtained from KURABO) have been grown in 96-well plates at a cell density of 2 x 10 ⁴ cells using DMEM medium (taken from SIGMA) containing 0.5% young bovine serum (FBS). ). After 24 hours of planting, the cells were replaced with DMEM medium containing 0.5% FBS containing a predetermined concentration of each bird's nest sample, and cultured for 24 hours.

Also, the sample concentration is as follows:

(i) Concentration of enzyme decomposition products (0, 78.1, 312.5, 1250, 5000 μg/ml)

(ii) Concentration of hot water extract (0, 1.56, 6.25, 25, 100 μg/ml)

After the cultivation, a part of the liquid on the medium was recovered, and the amount of type I collagen in the supernatant was determined by ELISA.

It can be seen from the measurement results that the enzyme decomposition product of bird's nest can actively proliferate type I collagen of human skin dermal fibroblasts. Compared with the hot water extract of the comparative example, the difference is remarkable.

After culturing, in a Hank's buffer containing a predetermined concentration of each bird's nest sample, after UVB (medium wave ultraviolet ray) is irradiated (using an ultraviolet illuminator (TL20W/12RS by PHILIPS), a UV short-wavelength cut filter ( The cut-off wavelength was 300 nm)), replaced with DMEM medium containing 0.5% FBS, and cultured for another 24 hours.

After the culture, the medium was recovered and the amount of type I collagen in the supernatant was determined by ELISA.

After dissolving the cells in 0.1% Triton X-100 solution, the amount of protein was quantified.

The ELISA method uses Anti-Human Collagen Type 1 antibody (Rabit) as a primary antibody, and uses Histofine PO (Rabit) as a secondary antibody, and the color former uses 2,2'-azino-bis-(3- Ethyl benzodiazepine-6-sulfonic acid) (ABTS), and the absorbance at 405 nm was measured by a microplate reader.

The quantification of the protein quality is carried out by the BCA method (di-cinchonine method) (a method in which a copper ion which has reacted with a peptide bond is reacted with BCA (dioctocinic acid) to measure the absorption at a wavelength of 562 nm).

The amount of type I collagen in the medium was calculated by dividing the amount of type I collagen in the medium by the amount of protein in all the cells to calculate the amount of type I collagen produced per unit amount of protein.

The result is shown in Figure 3.

It has been confirmed from the results that the enzyme decomposition product of bird's nest can actively suppress the decrease of type I collagen of dermal fibroblasts caused by UVB irradiation. Further, the hot water extract (comparative example) did not exhibit such an inhibitory effect.

Figure 1 is a graph showing the results of determination of the amount of citric acid in a bird's nest sample.

Figure 2 is a graph showing the results of the measurement of the amount of protein in the bird's nest samples.

Fig. 3 is a graph showing the results of measurement of the bird's nest enzyme decomposition product 1 by GPC-HPLC.

Fig. 4 is a graph showing the results of measurement of the bird's nest enzyme decomposition product 2 by GPC-HPLC.

Fig. 5 is a graph showing the results of measurement of the ability to promote collagen production.

Claims (11)

The use of a decomposition product obtained by pancreatic enzymatic decomposition of bird's nest is a decomposition product obtained by pancreatic enzymatic decomposition of bird's nest for producing a skin external composition for inhibiting the production of collagen, and the collagen Protein production is caused by ultraviolet light exposure of dermal fibroblasts of mammals including humans. The use of the first aspect of the patent application, wherein the dermal fibroblasts are irradiated by ultraviolet rays to cause a decrease in the production of collagen, and the inhibition of type I collagen production in skin dermal fibroblasts caused by ultraviolet irradiation is suppressed. . The use of the skin external application composition, wherein the skin external composition is used for a skin care device before or after receiving ultraviolet radiation. The use of the skin external composition according to the first aspect of the invention, wherein the skin external composition is used for preventing or improving the reduction of skin elasticity and gloss which occurs in the ultraviolet-irradiated skin and preventing or improving wrinkles. For example, the use of the above-mentioned enzyme decomposition product has a molecular weight of 70 to 200,000. For example, the use of the above-mentioned enzyme decomposition product has an average molecular weight of 1000 to 50,000. For example, the use of the skin external composition is a cosmetic. A method for inhibiting the production of collagen, which is caused by ultraviolet irradiation of low-grade dermal fibroblasts, comprising: decomposition of an effective amount of bird's nest by trypsinization On the skin of a mammal, including humans. The method of claim 8, wherein the dermal fibroblasts are irradiated with ultraviolet rays to cause a decrease in collagen production, and the ultraviolet ray irradiation causes a decrease in type I collagen production in skin dermal fibroblasts. . The method of claim 8, wherein the method is for skin care of the skin before or after exposure to ultraviolet light. The method of claim 8 is for improving skin elasticity and gloss, and preventing or improving wrinkles, and the skin elasticity and gloss are caused by ultraviolet rays.